Preliminary Results

 

Overview:

    We present preliminary data which support two points:  First, that as an experimental system S. tropicalis is virtually interchangeable with X. laevis with respect to basic embryology, development, and the application of advanced surgical,  genetic, and molecular manipulations and including in situ hybridization analyses using X. laevis probes and production of transgenic embryos and gynogenetic diploids.  This suggests that the activation energy required prior to performing advanced embryological analyses will be low.  Re-cloning most X. laevis genes from S. tropicalis to use as probes appears to be unneccessary; adaptation of most other techniques is straightforward.
 
    Second, that as a genetic system S. tropicalis, with its diploid genome and short generation time, evades X. laevis disadvantages and that in important practical respects compares favorably to the two current predominant vertebrate genetic models, mice and zebrafish.
 

Husbandry:

Our basic protocol for mating S. tropicalis adults and raising tadpoles is similar to that used for X. laevis with adjustments for S. tropicalisí smaller size and higher temperature optima (22-30oC vs. 16-22oC). Using this protocol we have been able to obtain fertile eggs from females as young as 4 months of age. The use of flow-through tanks to provide continuous circulation (which also greatly reduces husbandry labor costs), a richer froglet diet supplemented with brine shrimp and/or X. laevis tadpoles, and the use of hormonal treatments promise to reduce generation time even further (see specific aim 1.A). Sexual maturity at 3 months seems well within reach based on comparable husbandry optimization in X. laevis 59(Reinschmidt pers. comm.)  See Husbandry.

Cryopreservation of sperm:
    Protocols for long-term storage of viable sperm have been developed in X. laevis, making it possible to analyze mutant phenotypes at convenient intervals without the expense of carrying large numbers of heterozygous lines (D. Reinschmidt & R. Tompkins, pers. comm.). Thawed sperm motility is approximately 5-10% of unfrozen motility, but can effectively fertilize eggs.

Stock management:
    Pipid frogs are quite long-lived (X. gilli over fifteen years old have been bred, and X. laevis lifespan exceeds twenty years 27).  Marking unique animals should permit inexpensive housing in mixed groups for considerable long-term savings.  Individual frogs can be semi-permanently identified by branding anaesthetized animals with brass wire dipped in liquid nitrogen; brands on X. laevis last at least two years 59. Branding is inexpensive and convenient, and simple brands can be made on immature frogs.  We are using an unambiguous 20 character alphanumeric code; adult frogs can be branded with at least four characters, providing ample unique identifiers (160,000).
    Our S. tropicalis stock database system is modeled on the zebrafish databases in use at the University of Oregon 60, using Filemaker Pro III software (Claris Corporation, Santa Clara, Ca.). Fields recorded for each tank, spawning (sib-group), or identified individual include tank location; birth date; genotype (e.g. parental genotype, transgenic inserts, gynogenesis, haploidy, (potential) hetero/homozygous carrier of a mutation); #males, #females, #unsexed in the tank; mortality pre- and post- metamorphosis and identified causes; dates used for mating;  mating success; and, for individually-identified frogs of particular interest, a brand code.  See Grainger/Keller facilities.
 

Embryonic Development of S. tropicalis

Normal tables:
    Development of the S. tropicalis embryo differs from  X. laevis in only minor ways, aside from its smaller size (about half the diameter of  X. laevis (compare Fig. 1A and 1B in Figure 1), higher temperature optima, and slightly faster rate of development when cultured at the same temperatures. At 23-24 oC, onset of first cleavage occurs at 60', rather than 75' as in X. laevis.  Gastrulation is marked in both by the first appearance of concentrated pigment at the dorsal blastopore lip (Plate I Fig. 1A, B; arrowhead denotes dorsal lip of the blastopore) and begins in about 7 hours, compared to just under 9 hours for X. laevis, and procedes similarly.  Dorsal views of the late gastrula-early neurula (Fig. 1C,D, Figure 1) and late neurula (Fig 1E,F, Figure 1; arrow points to neural fold) are similar in the appearance, size, and development of the neural plate. Near the end of neurulation, the closure of the anterior neural tube differs in the more obvious constrictions of bottle cell apices.  Eye primordia at subsequent stages are more pronounced and situated slightly more ventrally than in X. laevis.  Internal development is also slightly altered, with the animalward migration of the head mesoderm and the yolky endoermal cells that constitute the floor of the blastocoel being more pronounced and occuring earlier than in Xenopus. Tadpoles are similar, though again  X. laevis is larger (Fig. 1G,H, Figure 1).
    S. tropicalis embryos differ from those of X. laevis in certain practical respects. Based on over twenty years of detailed analysis of morphogenesis of X. laevis and several years of analysis of S. tropicalis, we have found that the latter shows less variation in development from spawning to spawning and embryo to embryo, and a more predictable relationship between external staging criteria and internal development than the former. For example, as the bottle cells form in S. tropicalis, marking the onset of gastrulation at stage 10- (Fig. 2A, Figure 2:  arrowhead denotes dorsal lip), the internal view shows a consistent and predictable rolling upward of the involuting material on the dorsal side (Fig. 2B; arrowhead denotes dorsal lip). Slightly later, at stage 10+ (Fig. 2C, arrowhead denotes dorsal lip), the involution has occurred laterally and ventrally in all embryos (arrowheads in Fig. 2D, E; large arrowhead on dorsal side), whereas in X. laevis the progress of involution is less predictable. In X. laevis at stage 10- (Fig. 2F, arrowhead denotes dorsal lip) the mesoderm has generally not attached to the overlying ectoderm, but in some cases it has (arrow, Fig. 2G). Moreover, from a practical experimental perspective, X. laevis cells are sticky and hard to cut; tissue is often brittle, and fractures easily under microsurgical manipulation (pointers, Fig. 2G), whereas this is uncommon in S. tropicalisX. laevis also shows frequent defects in formation of the boundary of the vegetal endoderm and marginal zone, indicated by lack of bottle cell formation (arrow, Fig. 2H), and large variations in the amount of yolky, vegetal endoderm, and therefore the position of the involuting marginal zone ranges from far animally (pointers, Fig. 2I) to near the vegetal pole (pointers Fig. 2J). Neither of these extremes gastrulate normally.

Embryological Manipulations:

Explants of axial tissues:

Several types of early gastrula explants used to analyze induction, patterning and morphogenesis of axial tissues in X. laevis49, 61-63 have been made with S. tropicalis embryos, with similar development and results. The standard or "Keller" sandwich explant, consisting of two dorsal 120 degree sectors of the early gastrula sandwiched together (Fig. 3A, Figure 3) shows convergent extension of the mesodermal/endodermal (arrows, Fig. B,C) and neural (pointers, , Fig. B,C) components, as in X. laevis63(Fig. 3D). "Open-faced" explants, which expose the deep cells of the gastrula to observation and experimental methods, have been used to characterize the cell motility driving the convergent extension and patterning of the dorsal mesoderm64-66. In S. tropicalis, these explants show similar contrast and resolution of cell boundaries in epi-illumination (Fig. 3E), develop as well or better than their X. laevis counterparts, and are easier to make, due to the better cutting characteristics of S. tropicalis tissues. Biomechanical assays performed on explants of X. laevis67 could be readily adapted to S. tropicalis to support genetic analyses. In summary, the embryological preparations that have been most useful in resolving the cell behaviors, the biomechanics, and the tissue interactions important in embryonic development of X. laevis, can also be done with S. tropicalis with equal or greater ease and with the same productivity.

Prospective lens ectoderm transplants:
    One significant experimental advantage of amphibia is their tolerance of the transplantation of whole tissues from one embryo to another.  This can be invaluable in defining when and where signals are transmitted during inductive tissue interactions.  For example, in the eye, lens is instructed to form by signals from adjacent presumptive retina.  Transplantation assays in X. laevis have shown that ectoderm can only respond to these signals during a short period in development.  We repeated these assays in the smaller S. tropicalis to establish that this species was also amenable to these types of manipulations.
    Lens-competent, gastrula stage ectoderm from a fluorescein-dextran-labeled donor embryo was transplanted to the presumptive lens-forming region (PLR) of a neural plate stage host68, 69. As in X. laevis, labeled donor ectoderm went on to form a lens in roughly 50% (4 of 9) of the cases.   The host embryo and site of transplanted ectoderm (viewed by fluorescence microscopy) at the end of the experiment are shown in Fig 3F and G, respectively, in Plate I.  Sections through the head at this stage (Fig. 3H) show the donor tissue labelling (Fig. 3I).  The tissue identity of the transplanted lens was confirmed by staining with an antibody to X. laevis  ?-crystallin, an abundant lens protein (Fig. 3J).  This experiment suggests that embryological techniques developed in X. laevis are readily adapted to S. tropicalis.  Neither the slightly smaller size of the embryo nor the slightly higher temperatures required for development posed a significant impediment to the transplant procedure.

 

Analysis of Gene Expression:
    As S. tropicalis is closely related evolutionarily to X. laevis, it is possible to assay for the expression of many specific gene products using probes developed in X. laevis (see below).  If carefully designed, other assays should permit distinguishing between S. tropicalis and X. laevis gene products.  For instance, it is often useful to ask whether injection of a gene product induces its own expression, but induced mRNA is masked by input experimental message.  Rnase protection or RT-PCR assays, especially targeted to fast-evolving untranslated regions of mRNA, should facilitate detection of endogenous S. tropicalis mRNAs in the presence of excess injected X. laevis mRNA.

In situ hybridization:
    A wide variety of identified genes with tissue-specific expression patterns are used in X. laevis as molecular markers in of development and differentiation. Application of such markers to a new system is essential, but the effort in reisolating S. tropicalis equivalents is considerable. We have assayed a panel of X. laevis RNA probes for their ability to recognize cognate S. tropicalis  genes by whole-mount in situ hybridization. All of the X. laevis probes which have been assayed to date (the transcription factors Engrailed-270, Brachyury71, Pax-672, Six-3, Sox-3 (Zygar and Grainger, unpub.) and the lens protein ?-crystallin73 are shown in Fig. 4A-F, respectively) reproduce expression patterns in S. tropicalis that are indistinguishable from their expression in laevis. These results suggest that re-cloning S. tropicalis genes may not be required for many types of assays, and that overall nucleotide sequence and expression patterns may be very similar in both species.

Immunohistochemistry:
    Whole mount antibody staining is another useful technique which is readily adapted to S. tropicalis from X. laevis protocols.  We have assayed two antibodies, 12/101 (Fig. 4G), which specifically stains somitic mesoderm, and 6F11 (Fig. 4H), which recognizes Neural Cell Adhesion Molecule (NCAM) and stains brain, neural tube, and eye.  Both of these antibodies gave an identical staining pattern in S. tropicalis as in X. laevis.
 

Genomic Manipulations

Haploid and gynogenetic diploid S. tropicalis and X. laevis:
    A series of useful techniques have been developed in amphibians for manipulating the ploidy of embryos74-77.  Haploids develop fairly well before arresting at mid-tadpole stages.  By using UV-irradiated sperm to produce haploid embryos and then immediately restoring diploidy, it is possible to generate embryos that are homozygous over some or all of their genome in a single generation.  These techniques are likely to be especially useful in conjunction with transgenic procedures, when it will often be helpful to rapidly create lines of frogs that are homozygous for an introduced reporter locus or gene trap.  Production of gynogenetic diploids can also be used to uncover recessive mutations.  One study, using eight wild-caught X. laevis females, revealed twelve heritable developmental abnormalities74.  We plan to use this procedure for two principal purposes: to create healthy isogenic lines of S. tropicalis for improved experimental consistency, and to identify mutations carried by wild-caught animals to use as markers in subsequent genetic analyses.
We assayed gynogenesis by fertilizing albino X. laevis eggs with UV-irradiated wildtype sperm; at tadpole stages, haploid embryos remain unpigmented in contrast to embryos fertilized with untreated sperm (Fig. 5A, Plate II), demonstrating that UV-inactivation of the paternal genome was effective.  Haploid embryos develop a distinct phenotype, usually including axis kinking and ventral edema, but at early tailbud stages are virtually indistinguishable from diploid embryos and can be used as a background on which to uncover recessive mutations (Fig. 5B, Plate II).
    Restoration of diploidy to haploid amphibian embryos has been accomplished in several ways: heat shock; cold shock; early pressure to block second meiosis; and ìlate pressureî to block the first mitotic cleavage.  The pressure method has been most extensively characterized in pipid frogs77.  ìLate pressureî applied to haploid embryos results in embryos that are homozygous at all loci, and offers a method of rapidly creating truly isogenic strains.  ìEarly pressureî involves application of ~5000 lbs/in2 5-10 minutes after fertilization, and is thought to suppress the formation of the second polar body, which usually occurs 15 minutes after fertilization78.  Application of early pressure to haploids does not neccessarily result in embryos that are completely homozygous, due to meiotic crossover, but loci that are not distal to the centromere are likely to have been rendered homozygous.  We have used early pressure to successfully rescue haploid X. laevis (Fig. 5C; Plate II) and S. tropicalis in a high percentage of embryos (Fig. 5F, Plate II) (compare wildtype and haploid embryos in Fig. 5D and E, respectively, in Plate II).

Transgenic frogs:
    We recently developed a simple procedure for generating transgenic X. laevis in large numbers1;2. In Fig. 6A and 6B are shown brightfield and fluorescent images of living embryos expressing the cardiac muscle promoter driving GFP expression (from Kroll and Amaya, 1996). Integration of DNA into the chromosomes of the nuclei is very efficient using this procedure, such that greater than 50% of the resulting embryos are transgenic.  By using an infusion pump that delivers volume at a rate of 10nl/sec, two people are able to transplant over a thousand nuclei within a few hours.  The eggs are then screened for normal cleavage patterns signifying that the eggs received one nucleus.  Typically 20% to 30% of the transplanted eggs cleave normally, and as mentioned previously, most of these are transgenic.  One person can easily generate hundreds of transgenic embryos in a few hours.  Two people working together typically generate 400-600 viable transgenic embryos in an afternoon.
    During the course of preparing transgenic embryos, occasional examples expressed the reporter with aberrant, but highly regionalized tissue specificity.  In Fig. 6E-G are seen what are believed to be ìenhancer trapsî resulting from insertion of GFP constructs into chromosomal sites where reporter expression became controlled by a nearby enhancer.  Such fortuitous enhancer traps may be of great use as reporter lines in assaying embryological responses or in genetic screens (discussed further in Specific Aim 2B).
Several aspects of this procedure continue to be optimized by the Xenopus community, including the groups of Enrique Amaya and Rob Grainger. Decondensed sperm nuclei are very fragile, and transplantation of damaged nuclei results in abnormal development.  We handle the nuclei very carefully, and use very large needles (inner diameter of ~80 mm) for transplantation in X. laevis to minimize shear. As is the case in mouse transgenesis, high quality and purity of DNA minimizes toxicity to the embryos.

Transgenic S. tropicalis:
    The transgenesis procedure developed in X. laevis has been shown to be effective in S. tropicalis.  Figures 6H and I, Plate II,  show a transgenic stage tadpole expressing GFP tissue-specifically under the control of the X. laevis cardiac actin promoter. In preliminary optimization for S. tropicalis, it became evident that spermidine and spermine in the injection buffer is toxic to the embryos.  A buffer was designed to more closely approximate intracellular ion concentrations.  Our preliminary results with this buffer have been very encouraging, with 30% of the injected embryos going on to cleave normally. The fact that we have successfully produced S. tropicalis transgenic animals in so few attempts suggests that adapting the X. laevis protocol to S. tropicalis will not be problematic.

Characterized promoters:
    Since the transgenesis procedure is a recent development, most promoter analysis in Xenopus has been done with injected plasmids.  Expression from plasmids often does not reproduce tissue specificity of the promoter.  For example, the neural specific beta-tubulin promoter is expressed mosaically and clearly outside its domain of endogenous expression (differentiating neurons) (P. Krieg, pers. comm.).  The same construct reproduces the expected neuronal expression pattern when assayed in transgenic embryos (Fig. 6D, Plate II)1.  Other isolated promoter constructs that have been tested and work well in transgenics are the cardiac actin promoter1, the heart specific myosin light chain 2 promoter (Cooper, Mohun and Amaya, unpublished), the goosecoid promoter (Amaya and Cho, unpublished), and the XFKH1 promoter (Drayton, Amaya and Hill, unpublished).  Another promoter that we have begun to analyze is the brachyury promoter, which has been cloned by Ken Cho's group at UC Irvine.  We have also tested constructs containing 1.6kb upstream sequences of the brachury promoter in transgenic embryos (Amaya and Cho, unpublished).  Instead of getting expression through the marginal zone at the mid-gastrula stages, the notochord at the late gastrula and early neurula and the tailbud in later embryos (as would be expected from the endogenous gene expression pattern), this promoter construct drives strong expression in lateral and ventral marginal zone at the gastrula stage, but lacks dorsal and notochord components (Fig. 6C, plate II).  Furthermore, this construct maintains expression in the posterior somites in later embryos. We are currently searching for the dorsal-notochord enhancer elements as well as the repressor elements that turn off expression in the somites after gastrulation.  Our experiments with the brachyury promoter clearly show the value of using transgenic embryos to analyze promoter elements, since much information can be gathered about the spatial and temporal regulation of promoter elements in the context of the whole embryos with a small investment of time.

Euploid Xenopus cell lines:
    Tissue culture cells are valuable both as a simple model system for certain processes, and because very large numbers of individual genomes may be manipulated and selected for specific properties in vitro. Xenopus cell lines have been established from both embryonic and adult tissue explants79. However, long-term culture often leads to chromosomal loss, truncation, or translocation (aneuploidy); aneuploid cells may not reproduce all relevant biological functions, especially in nuclear transfer assays.

    We have established two new X. laevis cell lines (S3 1 and S1-2), both of which are derived from dorsal explants of late neurula embryos.  These cell lines are now clonal and have been in culture since 8-1-95; one of them is fibroblastic in morphology; the other epithelial.  Karyotype analysis suggests that both of these clonal cell lines are euploid. The karyotype of a cell of a non-clonal primary cell culture (Fig. 7A, Plate II) shows an aneuploid chromosome number (Fig. 7B, Plate II), while the cell line S3-1 (Fig. 7C, Plate II) shows a euploid karyotype (Fig. 7D, Plate II). More than 60% of the mitotic nuclei in both clonal cell lines assayed showed thirty-six chromosomes with no visible abnormalities.  The fact that we can establish euploid X. laevis cell lines in culture suggests that generation of similar S. tropicalis lines is feasible.